Stimulation of Active Uptake of Nucleosides and Amino Acids by Cyclic Adenosine 3’:5’-Monophosphate in the Yeast Schizosaccharomyces porn be*

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3’:5’-monophosphate to Schizosaccharomyces pombe 972h-wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cereuisiae strain IL 216-IA. The mediated uptake of 2-n-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,02’-Dibutyryl adenosine 3’:5’-monophosphate is also efficient, whereas theophylline, guanosine 3’:5’-monophosphate, 5’-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without


Stimulation
of Active Uptake of Nucleosides and Amino Acids by Cyclic Adenosine 3':5'-Monophosphate in the Yeast Schizosaccharomyces porn be* (Received for publication, July 18, 1974) FRANCOISE FOURY AND ANDRE GOFFEAU From the Laboratoire d'Enzymologie, Groupe EURATOM, Universitt! de Louvain, Place du Sud, 1, Sciences 14Bj3, 1348 Louvain-La-Neuve, Belgium In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h-wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cereuisiae strain IL 216-IA. The mediated uptake of 2-n-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,02'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport and cellular ATP level, indicating that the ATP required for transport is not necessarily of mitochondrial origin. The uptake of 2-n-deoxyglucose is unaffected by the respiratory inhibitors.
Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake.
In the absence of respiration, Dio-9, an ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport.
In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venturicidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate.
These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-g-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.
Cyclic adenosine 3':5'-monophosphate modifies the trans-cyclic AMP' was reported to interfere with the phosphorylation port of ions, amino acids, and sugars across the plasma of a specific membrane protein which might be involved in the membrane of several animal tissues (l-6). It has been proposed control of cellular permeability (8,9). Modification of memthat such stimulations of ion fluxes are related to stimulations of glycogenolysis and gluconeogenesis (2,7). More recently, 'The abbreviations used are: cyclic AMP, cyclic adenosine brane permeability by cyclic AMP was also demonstrated in cell cultures (10-12). High intracellular cyclic AMP concentrations produced by serum deprivation in normal fibroblasts exert a negative control on growth and transport, possibly at the microtubule aggregation level (11). The regulatory processes, abolished in malignant transformed cells, can be partly restored by addition of exogenous cyclic AMP (10).
Control of cellular permeability by cyclic AMP has received much more attention in mammals than in yeast and in bacteria. Yeast, however, is a particularly suitable organism. It contains adenosine 3':5'-monophosphate, a specific phosphodiesterase, adenylyl cyclase, and a cyclic AMP-dependent protein kinase (13-17). Elaborate genetic techniques are available and the results obtained with this simple eukaryote are more likely to be extended to mammalian cells than those obtained with bacteria. A convenient approach to study the role of cyclic AMP in bacteria as well as in fibroblasts has been the addition of the cyclic nucleotide to the culture medium (10, 18). Unfortunately, until now, few significant effects of exogenous cyclic AMP have been reported in yeast (19,20). In a previous paper (21), we reported that exogenous cyclic AMP stimulates the incorporation of uridine into RNA in a glucose-superrepressed mutant COB5 (22) of the yeast Schizosaccharomyces pombe subjected to glucose deprivation.
In the present paper, we show that this stimulation is most likely the result of a stimulated transport of uridine across the plasma membrane. Characterization of the active uptakes of nucleosides and of amino acids in several yeast strains under starvation conditions leads us to suggest a possible role of ATP and of a plasma membrane ATPase in the cyclic AMP-stimulated active transport of yeast.

Culture Conditions
The YD medium contains 58 g of glucose and 20 g of yeast extract (Difco) per liter of water. The YDG medium contains 36 g of glycerol, 1 g of glucose, and 20 g of yeast extract per liter of water. Both media were brought to pH 4.5 with HCl. Cultures were inoculated with 10" cells per ml from an overnight preculture and grown at 30" with vigorous shaking. Cells were harvested in the exponential phase of growth by centrifugation at 4' and washed with cold sterile water only when indicated.

Metabolite
Uptake Measurements Immediately after harvest, the cells were transferred into the starvation medium containing 20 mM Tris adjusted with citric acid to pH 4.5. Then, 5 x lo7 cells per ml were preincubated for 15 min in the presence or absence of 1 mM cyclic AMP before introduction of the labeled compound. Incubations were carried out at 30" with continuous shaking. At the indicated times, samples of 2.5 x 10' cells were diluted into 10 volumes of cold water and collected on Gelman filters (0.8 pm pore size) prewashed with 0.5% of the unlabeled compound. The cells were immediately washed with 20 ml of cold water and dried. The filters were introduced into glass vials containing 7 ml of a toluene mixture (21) and counted with a scintillation spectrometer. Incorporations of [5-aH]uridine into RNA and of L-[U-"Clleucine into proteins were measured as previously described (21).

Cellular cyclic AMP Measurements
Three grams of glycerol-grown cells (wet weight) were extracted with 6 ml of 10% cold trichloroacetic acid and ground for 2 min with 5 g of glass beads (0.45 pm diameter) in a Braun homogenizer at about 4". The supernatant was extracted five times with 2 volumes of diethyl ether to remove trichloroacetic acid and lipids. Ether was evaporated by boiling the extract for 2 min at loo", and the pH was brought to 6.5 with NaOH. One milliliter of the extract containing a trace of cyclic [G-'HIAMP to estimate the cyclic AMP recovery was purified by chromatography on a Dowex 5OW-X4 column (5 cm high and 5 mm diameter) under 120 cm of H,O pressure. Elution was carried out with water. The cyclic AMP cellular content was then determined by Gilman's method (25) with a Boehringer kit. A Dowex blank was carried out according to Otten et al. (26). The results were identical with those obtained by the method of Van den Berg et al. (27).

Cellular ATP Measurements
Five hundred milligrams of cells (wet weight) were collected on Millipore filters (0.8 lrn pore size and 47 mm diameter) at 2" and immediately dipped into 2 ml of 10% cold trichloroacetic acid. After 15 hours of trichloroacetic extraction, the cells were sonicated for 2 min and centrifuged (28). The supernatant was treated with diethyl ether as described above. The ATP content was evaluated by spectrophotometric measurements of NADPH produced by the addition of 15 mM glucose to the incubation medium containing 8 mM MgCl,, 0.1 mM NADP, 1 unit of glucose-6-dehydrogenase and 7 units of hexokinase in 100 mM Tris-HCI, pH 8.0. The cellular glucose-6 phosphate content was substracted.

Cellular Amino Acid Pool Measurements
First, 4 x 10Q cells were incubated at 0" in 2 ml of trichloroacetic acid for 1 hour. After centrifugation, the supernatant was treated with diethyl ether as described above and was evaporated. The samples were dissolved in 0.2 M sodium citrate buffer, pH 2.2, and aliquots were applied to a Hocaste amino acid analyzer (Hocaste Co, London). Analysis was carried out with a single column system using stepwise elution.

Antibiotics and Inhibitors-Antimycin
A is from Boehringer; anisomycin is from Pfizer Laboratories; cycloheximide is from Sigma; carbonylcyanide m-chlorophenylhydrozone is from Calbiochem; N,N'dicyclohexylcarbodiimide is from British Drug House Laboratories; Dio-9 was purchased from Koninklijke Nederlandsche Gist and Spiritus fabriek, Delft, The Netherlands; venturicidin A was a gift from Dr. J. Mattoon, Johns Hopkins University of Baltimore. When the inhibitor is diluted in ethanol, an equal volume of ethanol is added to the control.

Stimulation
of &dine and L-Leucine Uptakes by Cyclic AMP-Glycerol-grown cells of Schizosaccharomyces pombe COB5 were collected in the exponential phase of growth and transferred into a buffer without glucose and nitrogen. In this 'The performance of amino acid analyses by Dr. R. Crighton is gratefully acknowledged. condition of starvation, the uptake of added uridine was linear for more than 5 min and markedly stimulated within 30 s by the addition of 1 mM adenosine 3':5'-monophosphate ( Fig. 1A). A slow incorporation of uridine into RNA began 3 min after the uptake. This incorporation which accounts for only 10% of the total uptake was also enhanced by cyclic AMP. Similar stimulations were obtained for L-leucine uptake and incorporation (Fig. 1B). These results suggest that cyclic AMP acts primarily on the cell membrane transport. However, a slight but distinct effect on RNA synthesis itself cannot be excluded. The stimulation of transport might be due to a stimulated metabolization of uridine or L-leucine, eliciting a decreased internal pool and reduced feed-back control of the uptake (29). The intracellular amino acid pool however is not significantly modified during the 20-min incubation in the presence of cyclic AMP (Table I). Moreover, we shall see below that the uptake of oc-aminoisobutyric acid, a nonmetabolized amino acid, is also enhanced by cyclic AMP (Table II). We can therefore conclude that cyclic AMP stimulates the uptake process itself, and that the increased incorporations of uridine into RNA, and of L-leucine into proteins are mainly the result of stimulated transport across the cell membrane.
Cyclic AMP Specificity-W, 0"-Dibutyryl cyclic AMP is commonly used instead of adenosine 3':5'-monophosphate, as it is more efficient in fibroblasts and mammals. The main reason for this higher efficiency is that N6,02'-dibutyryl cyclic AMP inhibits cyclic AMP phosphodiesterase and prevents the intracellular cyclic AMP degradation, just as theophylline does (30). In S. pombe however, we do not find any difference of efficiency between the two cyclic nucleotides (Fig. 2). Theophylline (1 mM to 20 mM) even in the presence of exogenous cyclic AMP, does not affect the transport (data not shown). Whether exogenous theophylline prevents cyclic AMP degradation in S. pombe is unknown, but we must note that the  Glycerol-grown cells of S. pombe COB5 were harvested ia the exponential phase of growth and transferred into the starvation medium, containing 1 mM cyclic AMP when indicated. The cells were incubated at 30" for 30 min and collected on Millipore filters as described in "Material and Methods." Ten minutes before the end of incubation, antimycin A (final concentration 10 PM) was added to a sample incubated without cyclic AMP.
Amino acids Cells (5 x 107) in 1 ml were first preincubated for 15 min in the starvation medium containing 1 mM cyclic AMP, when indicated the cells were incubated for 5 min with 5 kCi of [2,8-3H]adenosine, [8-3H]guanosine, [5-3H]uridine, L- [2,3-3H]aspartic acid, [2-3H]  reported in mammals. In kidney cortex, cyclic AMP stimulates the V,,, of cu-methylglucoside uptake, whereas it decreases the apparent K, of amino acid entry in jejunal mucosa (5,6). Our results show that in S. pombe the maximum velocities of the uptakes of metabolites as different as uridine, amino acids, and sucrose, most likely mediated by distinct carriers, are all stimulated by cyclic AMP. Interaction of cyclic AMP at the level of a specific carrier can thus be excluded. Because the apparent affinities of the metabolite uptakes are not modified, a direct effect of the cyclic nucleotide on the binding of a general "carrier system" is rather unlikely.
Stimulation of Uptake by Cyclic AMP Requires Both Glucose Deprivation and Intracellular ATP-All of the preceding experiments were performed with glucose-starved yeast cells. In these conditions, the uptake is low and markedly enhanced by cyclic AMP. Increasing the glucose concentrations from 0 to 10 mM in the incubation medium enhances the uptake of uridine up to a plateau (Fig. 4A). The extent of stimulation by cyclic AMP is most pronounced in the absence of glucose, and gradually decreases and finally disappears at higher glucose concentrations (Fig. 4A). Starvation conditions are therefore required for obtaining the stimulation of uptake by cyclic AMP.
During the incubation of resting cells in the starvation medium, ATP continues to be produced by an active endogenous respiration and its intracellular content remains as high as in growing cells (Table III). The addition of respiratory inhibitors such as antimycin A and of oxidative phosphorylation inhibitors such as NJ'-dicyclohexylcarbodiimide or Dio-9 (34, 35) leads to a drastic fall in the cellular ATP level with a concomitant decrease of uridine uptake and disappearance of the stimulation by cyclic AMP (Table III and Table IV, The effects of exogenous cyclic AMP are thus more specific in S. pombe than in fibroblasts where other adenine derivatives interfere with membrane transport (10).
Strain Specificity and Growth Conditions-The stimulation of the uptake of uridine by cyclic AMP occurs only in certain strains and only under certain culture conditions. The uptake of uridine is stimulated by cyclic AMP in glycerol-grown cells of the wild strain S. pombe 972h-submitted to glucose starvation.
In similar conditions the cyclic nucleotide fails to increase the uptake in 19 different glycerol-grown strains of S. cereuisiae. Only the strain IL126-1A (a Ural-,p+w+ C"E;,,) was clearly sensitive to the cyclic nucleotide.
In the wild strain, S. pombe 972h-grown on 320 mM glucose and harvested either in exponential phase or in stationary phase of growth, a glucose shift down does not induce stimulation of uptake by cyclic AMP. Similarly, no stimulation is observed with three glucosegrown respiratory-deficient mutants, COB2, COB7, and M126. Curiously, in glucose-grown as well as in glycerol-grown cells, the uptake of uridine is stimulated by cyclic AMP both in the glucose-derepressed mutant of S. pombe COB6 and in the glucose-superrepressed mutant COB5 (22). Substrate Specificity-In fibroblasts, the uptake of leucine, uridine, and 2-n-deoxyglucose is inhibited by adenosine 3':5'monophosphate (10-12). In jejunal mucosa, only the transport of basic amino acids is enhanced by cyclic AMP (5), whereas in kidney cortex, the uptake of all of the amino acids is stimulated (4). It was therefore of interest to determine the effect of cyclic AMP on the uptake of various metabolites in S. pombe COB5 Under starvation conditions, the uptakes of glycine, five distinct L-amino acids, cY-aminoisobutyric acid, adenosine, uridine, sucrose, and 2-n-deoxyglucose show saturation kinetics (Fig. 3), each with a distinct K, (Table II). These transports are likely mediated by specific "carriers" as in other yeasts (31-33). The uptake of guanosine is very low, and its apparent K, is too high to be determined.
The rates of uptake of neutral, acidic and basic L-amino acids, cr-aminoisobutyric acid, adenosine, uridine, and sucrose are stimulated twice to three times by cyclic AMP, whereas the uptake of 2-n-deoxyglucose and guanosine is not modified. In all cases, cyclic AMP stimulates the maximum velocity of entry and does not modify the apparent K, (Table II) 10.0 Experiment 1). It can be concluded that uridine uptake and its stimulation by cyclic AMP measured in starvation conditions require a high cellular ATP content. This ATP is not necessarily of mitochondrial origin because in the absence of respiration inhibited by antimycin A, the addition of 2.5 mM glucose raises the level of ATP produced by glycolysis to a value similar to that of growing cells (Table III) and restores the stimulation of uridine uptake by cyclic AMP (Table IV, Experiment 2). Increase of the glucose concentration up to 20 to 100 mM does not increase further the ATP content (Table III) but enhances the rate of uridine uptake whereas the extent of stimulation is gradually reduced to zero (Fig. 4B). The cellular concentration of L-leucine is about 1000 times higher than that of the external medium in starvation conditions. The ATP-dependent uptake of amino acids is thus an active transport occurring against a concentration gradient.
On the other hand, the uptake of 2-n-deoxyglucose which is most likely a passive process down the concentration gradient (33) and does not seem to require a Therefore, the cyclic AMP-stimulated uptake is an active mediated transport occurring against a concentration gradient and requiring high cellular ATP concentrations.
In the absence of ATP, cyclic AMP per se is not able to induce active transport and does not produce any increase of the cellular steady state ATP level (Tables III and IV, Experiment  1). ATP requirement for uptakes of nucleosides, amino acids, and sucrose might have several causes. A high intracellular ATP level could generate a higher incorporation of precursors into macromolecules and thus decrease the internal amino acid or nucleoside pool and consequently the negative feed-back control. In the absence of glucose, antimycin A which blocks the respiration and dramatically decreases the intracellular ATP concentration (Table III) and transport (Table  IV, Experiment l), does not modify significantly the intracellular pool of any amino acid in S. pombe within a lo-min glucose starvation period (Table I). The main role of ATP therefore is not to reduce the negative feed-back control. It is more likely that ATP might be involved in the formation of a "rich energy" compound (36) or of a potential gradient participating to the active transport across the plasma membrane. The chemioosmotic Mitchell's hypothesis proposes that in microorganisms, the driving force for active transport is created by an electrochemical gradient (positive potential outside) generated across the plasma membrane by electrogenic extrusion of protons into the external medium (37, 38). It was suggested by Conway (39) that in yeast an oxidation-reduction pump is implicated in proton extrusion. More recently, it has been assumed that a plasma membrane ATPase is involved in the active extrusion of protons into the external medium (40).
Although several lines of evidence support this view in bacteria and fungi (41-44), little supporting evidences are available in yeast. The following experiments were carried out in order to investigate the effects of cyclic AMP on proton extrusion in S. pombe. Stimulation of Proton Extrusion by Cyclic AMP-COB5 cells were preincubated in the starvation medium in the presence or absence of 1 mM cyclic AMP for 15 min prior to the addition of 10 mM antimycin A to block the respiration and of 5 mM glucose to allow the cells to glycolyze (Fig. 5). After a lag of 1 min, protons were extruded at the rate of about 2 weq per min and per g of protein. A slight but reproducible increase of proton extrusion (about 0.5 weq per min and per g of protein) was produced by cyclic AMP. In the same conditions, the stimulation of L-leucine uptake by cyclic AMP (50 pM in the external medium) attained 0.1 pmol per min and per g of protein.
The stimulation of proton extrusion seems thus significant especially because the extruded protons were mea- sured after dilution in the large external volume and as the acidification might be considerably amplified in the space localized between the cell membrane and the wall. The proton-conducting agent carbonylcyanide-m chlorophenylhydrazone inhibited both transport (Table IV, Experiment 2) and proton extrusion. It did not ,however inhibit specifically the cyclic AMP-stimulated transport, suggesting that cyclic AMP does not produce the electrochemical gradient. A functional relationship between stimulations of active uptake by cyclic AMP and proton extrusion is suggested by their parallel appearance under different conditions. For instance, cyclic AMP fails to enhance both uptake and proton extrusion in glycerol-grown cells of S. pombe COB5 transferred into a medium containing 50 mM glucose, or in glucose-grown cells of the wild strain transferred into the starvation medium (Fig. 5). Inhibition of Uptake by Dio-9-Dio-9, an antibiotic of unknown structure, is a potent inhibitor of the ATPases of chloroplasts, yeast mitochondria, and Streptococcus faecalis, and does not significantly affect the production of glycolytic ATP (35,42,45). In rat liver mitochondria, Dio-9 inhibits oxidative phosphorylation (46). COB5 cells were incubated in the starvation medium for 10 min in the presence or absence of 1 mM cyclic AMP. Antimycin A was then added to block the respiration and glycolysis was induced by 2.5 mM glucose. In these conditions, stimulation of the uptake of uridine by cyclic AMP was observed. Further addition of 25 pg of Dio-9 per ml of cell suspension blocked instantaneously the uptake of uridine both in the control and in the cyclic AMP-treated cells ( Fig. 6 and Table IV, Experiment 2). The inhibition of uridine uptake by low concentrations of Dio-9 is more pronounced in cyclic AMP-treated cells; the apparent K, for Dio-9 is 3 wg per ml in the presence of cyclic AMP and 5 pg per ml in the control. Dio-9 also inhibits the extrusion of protons into the external medium (data not shown). In these experiments, the inhibition of uptake by Dio-9 is not the result of inhibition of mitochondrial ATP production, because prior to the addition of Dio-9, the cellular respiration was totally abolished by antimycin A. The intracellular ATP level is unaffected by Dio-9 in nonrespiring cells inhibited by antimycin A (Table III). In the same conditions, only high (possibly nonspecific) concentrations of NJ'-dicyclohexylcarbodiimide (1 mM), another ATPase inhibitor (45) are inhibitory (Table IV, Experiment  2). Venturicidin A, an inhibitor of the mitochondrial membrane-associated ATPase (47) is totally inactive in antimycin A-treated cells, although it has some inhibitory effects on the uptake of uridine in respiring cells (Table IV, Experiment  3). In addition, we found that the cyclic AMP-insensitive uptake of 2-o-deoxyglucose is not inhibited by Dio-9 in the presence of antimycin A (Table IV, Experiment 4). These findings suggest that cyclic AMP stimulates indirectly or directly a Dio-g-sensitive function of the plasma membrane involved in active transport. In S. faecalis, Dio-9 inhibits active K+ uptake most likely driven by a plasma membrane ATPase (43). Such an ATPase is likely present in yeast (48) and is also Dio-g-sensitive.

Synthesis
Inhibitors on Cyclic AMPstimulated Transport-Stimulation of uptake is best expressed after a 15-min preincubation of the cells in the presence of cyclic AMP. Protein synthesis occurring during such preincubation might be required for the expression of the cyclic AMP effect. We have therefore investigated the effect of protein synthesis inhibitors on the uptakes of leucine and uridine. Anisomycin and cyloheximide which block protein synthesis at the elongation step (49) decrease partially and equally the uptake of L-leucine in cyclic AMP-treated cells and in the control (Table V). The stimulation by cyclic AMP does not require newly synthesized proteins during starvation.
It must be noted that in these conditions, the leucine and total amino acid pool are decreased by less than 20%, making rather unlikely a negative feed-back control of the amino acid uptake.
On the other hand, anisomycin and cycloheximide only slightly inhibit the uptake of uridine in the control (Table V). Curiously, the extent of the stimulation is markedly decreased by cycloheximide but slightly enhanced by anisomycin. This result, possibly related to the stringent control of RNA synthesis and to the stimulation of uridine incorporation by cycloheximide that we have described in starved S. pombe COB5, deserves further investigations (21). Cycloheximide (250 pg) or anisomycin (10 pg) per ml of suspension containing 5 x lo7 cells were added at the beginning of the incubation in the starvation medium in the presence or absence of 1 mM cyclic AMP. Fifteen minutes later, 10 nmol of [5-3H]uridine (5 PCi) or 50 nmol of L-[U-"Clleucine (1 FCi) per ml of suspension were added to the medium. The incubation was further carried out for 5 min. In order to determine whether the stimulation of active transport by exogenous cyclic AMP reflects a prior physiological decrease in the intracellular cyclic AMP level during starvation, we measured the variations of the internal cyclic AMP concentration under different conditions. The concentration of adenosine 3':5'-monophosphate in glycerol-grown S. pombe COB5 harvested in the exponential phase of growth, calculated from four different experiments, is about 2.5 nmol per g of dry weight. In cells further incubated for 30 min or 1 hour in the starvation medium, the internal cyclic AMP concentration is decreased only by about 30% (Table VI). On the other hand, if the cells are incubated for the same period in the presence of 5 mM glucose, no decrease of the cellular cyclic AMP concentration can be detected. Can this slight cyclic AMP concentration decrease account for the drastic increase of metabolite uptake produced by the addition of cyclic AMP to the starvation medium? Our results are in agreement with the small variations of the cyclic AMP level found in fibroblasts or in jejunal mucosa under different physiological conditions (5,11). These data indicate either that the cyclic AMP target is located at the outside of the plasma membrane or that several cyclic AMP pools are present in the cell. DISCUSSION Our data show that exogenous adenosine 3':5'-monophosphate prevents the negative control exerted by glucose starvation on the uptake of a number of metabolites across the plasma membrane in resting cells of S. pombe COB5. In order to investigate the role of cyclic AMP in the active transport of glucosestarved yeast cells, it was necessary to study first active transport itself, a very poorly documented function in yeast resting cells.

Role of ATP
in Active Transport of Resting Cells-Our data show that in glucose-starved cells of S. pombe COB5, previously grown on glycerol, metabolites are actively transported. These resting cells contain about 10 nmol of ATP per mg of protein produced by high endogenous respiration. The addition of respiratory and oxidative phosphorylation inhibitors dramatically decreases both cellular ATP content and uptake. Further addition of 2.5 mM glucose to the incubation medium restores both cellular ATP content and active uptake. We found however that higher glucose concentrations enhance several times the rate of active transport but do not lead to any further increase of the steady state ATP level. These data do Glycerol-grown cells harvested in exponential phase of growth were incubated for 30 min in the absence of glucose and nitrogen or with 5 mM glucose (Experiment 1). In Experiment 2, cells were incubated for 1 hour in a medium containing 0.1% yeast extract (pH 4.5)